Antifungal composition

ABSTRACT

Use of a combination of the squalene epoxidase inhibitor terbinafine and an azole 14-alpha-methyldemethylase inhibitor (fluconazole or itraconazole) in mycotic infections caused by azole-resistant fungal strains, and corresponding pharmaceutical compositions, process and method.

This is a 371 of PCT/EP96/02022, filed May 10, 1996.

The present invention relates to the treatment of human mycoticinfections. It concerns antifungal compositions for use in the treatmentof mycotic infections caused by azole resistant yeast strains,comprising terbinafine with an azole 14α-methyldemethylase inhibitorsuch as the azole fluconazole and/or itraconazole.

Although they are often present as benign commensal organisms in thedigestive tract of healthy individuals, fungi, particularly Candidaspecies produce a broad range of serious illnesses in compromised hosts.Such infections are clearly on the rise. Oropharyngeal candidiasis isthe most common fungal infection in patients with human immunodeficiencyvirus (HIV) infection. With the introduction of azole antifungal agentsthat are bioavailable after oral administration, the approach to thetreatment of serious Candida infections is possible. Ketoconazole, thefirst of there agents to become available, was quickly found to beefficacious in the setting of chronic mucocutaneous candidiasis.However, not long after the introduction of this agent, clinical failurein association with elevated minimum inhibitory concentrations (MICs) ofketoconazole that developed during prolonged therapy were reported. Thisproblem achieved prominence with the subsequent introduction offluconazole. Fluconazole, a water-soluble triazole with greater than 90%bioavailability after oral administration, is used extensively to treata wide range of Candida infections. In particular, it is widely used astherapy for oropharyngeal candidiasis in patients with advanced HIVinfection and AIDS. Although oropharyngeal candidiasis usually respondsreadily to fluconazole, it is difficult to completely eradicate theinfection and relapse often occurs within several months following thecompletion of therapy. For this reason, many AIDS patients receivefluconazole either continuously on intermittently over long periods oftime.

To a greater extent than with other azoles, resistance to fluconazolehas developed and is becoming a significant clinical problem, asattested by isolation from, in particular, AIDS patients of numerousCandida strains showing resistance (see e.g. D. Law et al., J.Antimicrob. Chemother. 34 [1994] 659-668).

It has now been found that, surprisingly, a combination of the squaleneepoxidase inhibitor terbinafine (Lamisil®) and an azole14α-methyldemethylase inhibitor such as fluconazole and/or itraconazoleis active against azole-resistant fungal strains. By using thiscombination of compounds there is provided a method for treating humanmycotic infections caused by azole-resistant fungal strains.

Suitable azole 14α-methyldemethylase inhibitors are in particularimidazole and triazole antifungal agents.

Preferred imidazole antifungal agents include clotrimazole(Arzneim.-Forsch. 22 [1972] 1280), miconazole (Arzneim.-Forsch. 21[1971] 256; econazole (Arzneim.-Forsch. 25 [1975] 224); isoconazole(Arzneim.-Forsch. 29 [1979] 1344); trioconazole (Antimicrobial AgentsChemotherapy 15 [1979] 597-602); sulconazole (Eumycetes and Mycosis 23[1982] 314-317; oxiconazole (Arzneim.-Forsch. 32 [1982] 17-24);cloconazole (J. Med. Chem. 26 [1983] 768-770); bifonazole(Arzneim.-Forsch. 33 [1983] 517-524); butoconazole (J. Med. Chem. 21[1978] 840; fenticonazole (Arzneim.-Forsch. 31 [1981] 2127);zinoconazole (J. Med. Chem. 26 [1983] 442-445) and ketoconazole (J. Med.Chem. 22 [1979] 1003-1005).

Preferred triazole antifungal agents include terconazole (J. Med. Chem.26 [1983] 611-613); itraconazole (Antimicrobial Agents and Chemotherapy26 [1984] 5-9); vibunazole (Arzneim.-Forsch. 33 [1983] 546); fluconazole(Antimicrobial Agents and Chemotherapy 27 [1985] 815-818), and(R)(-)-α-(4-chlorophenyl)-α-(1-cyclopropyl-1-methylethyl)-1H-1,2,4-triazol-1-ethanolin free form or in salt or metal complex form (GB 2'161'483)(hereinafter briefly referred to, in free form, as "compound A").

Especially preferred azoles are itraconazole and fluconazole.

Not all combinations of antifungal drugs show synergistic or evenadditive effects, and even antagonistic effects have been reported inthe literature. Thus in e.g. E. Martin et al., Antimicr. Agents andChemother. 38 [1994] 1331-1338, it has been reported that fluconazoleantagonizes the candidacidal action of amphotericin B; in Abstr. Ann.Meeting Am. Soc. Microbiol. 87 (1987) 392 it was reported that the useof terbinafine and ketoconazole in Candida albicans produced noenhancement of antimycotic activity; in Eur. J. Clin. Microbiol. Infect.Dis. 7 (1988) 732-735 it was stated that in vitro terbinafine appears toact antagonistically with the azoles; and in Drugs Today 24 (1988)705-715 it is again mentioned that in combination therapy, not allcombinations of antifungal drugs show synergistic or even additiveeffects.

It appears therefore that combination therapy with antimycotics ishighly unpredictable.

Thus, while one might on theoretical grounds expect that combinations ofpharmacologically active agents that inhibit a single biosyntheticpathway at two separate steps are normally more active than those whichact only on one step, and that combinations of e.g. terbinafine withazoles such as fluconazole and/or itraconazole should possess at leastadditive activity, it is very surprising that, for unknown reasons, suchcombinations are still effective even where resistance to the azoles hasalready developed, namely, synergistic effects are maintained even insituations where the mycotic strains have become resistant to theazoles.

The composition of the invention for treating human mycotic infectionscaused by azole-resistant fungal strains comprises an azole14α-methyldemethylase inhibitor such as fluconazole and/or itraconazole,and the arylmethylamine squalene epoxidase inhibitor terbinafine offormula ##STR1## in free base or acid addition salt, e.g. hydrochlorideacid addition salt form.

The most preferred azole is fluconazole. Terbinafine preferably is inpharmaceutically acceptable, preferably in hydrochloride salt form. Thepreferred fungus preferably is a yeast, if preferably is of the genusCandida, it especially is Candida albicans. The azole-resistance inducedmycosis may be superficial or systemic, it especially is oropharyngeal.It is deleterious to e.g. the skin or mucosa.

The azole-resistance may be a cross-resistance and involve multipleazoles.

The antifungal composition of the present invention is prepared byincorporating the arylmethylamine of formula I to an azole14α-methyldemethylase inhibiting antifungal agent, such as fluconazoleand/or itraconazole.

The invention thus concerns an antifungal composition for use in thetreatment of mycotic infections caused by azole-resistant fungal strainsfor the avoidance or restriction of deleterious azole-resistance inducedmycosis, comprising terbinafine as defined above and an azole14α-methyldemethylase inhibitor, such as fluconazole and/oritraconazole.

It further concerns the use of an antifungal composition comprisingterbinafine as defined above and an azole 14α-methyldemethylaseinhibitor, such as fluconazole and/or itraconazole, in the preparationof a medicament for use in the treatment of mycotic infections caused byazole-resistant fungal strains for the avoidance or restriction ofdeleterious azole-resistance induced mycosis in a human subjectcompromised by such azole-resistance.

It further concerns a process for the preparation of an antifungalcomposition as defined above, comprising the incorporation ofterbinafine as defined above to an azole 14α-methyldemethylaseinhibiting antifungal agent, such as fluconazole and/or itraconazole.

It further concerns a method of treatment of an azole-resistant fungalinfection caused by azole-resistant fungal strains for the avoidance orrestriction of deleterious azole-resistance induced mycosis, comprisingadministering a therapeutically effective amount of an antifungalcomposition as defined above to a patient in need of such treatment.

In the antifungal composition of the present invention the weight ratioof the azole antifungal agent to the arylmethylamine antifungal agentmay be varied within a wide range, but is preferably within a range offrom 100:1 to 1:500, more preferably from 25:1 to 1:125. By mixing theazole antifungal agent and the arylmethylamine antifungal agentterbinafine in a weight ratio within the above range, an excellenteffect is obtainable in the treatment of mycosis caused by azoleresistant fungal strains, particularly yeast strains, e.g. Candidastrains such as Candida albicans, Candida (=Torulopsis) glabrata,Candida krusei and Candida tropicalis; Cryptococcus strains such asCryptococcus neoformans; or Trichophyton strains, e.g. Trichophytonmentagrophytes; especially Candida strains, particularly Candidaalbicans.

The composition of the present invention can be adapted for localadministration and can be incorporated in a usual pharmaceutical carrierin a wide range of concentrations (usually in an amount of from about0.1% to about 10% by weight of the total composition) to prepare aformulation. The composition of the present invention can be used fororal administration in the form of tablets, capsules or a liquid, and itmay also be used for non-oral administration such as subcutaneous,intramuscular or intravenous injection. It thus normally is a fixedcombination. However, administration of the active agents may also beeffected in the form of a free combination, i.e. separately, e.g.sequentially, in any order.

The beneficial activity can be shown in vitro using variousazole-resistant strains. The assay is performed with RPMI 1640 medium in96-well, flat-bottom microdilution plates using a chequered drugdilution system. Terbinafine (in hydrochloride salt form) and the azoleare used in concentrations from 100 μg/ml to 0.006 μg/ml. Minimalinhibitory concentrations (MIC) are determined after a 48-hourincubation at 37° C. Minimal fungicidal (MFC) concentrations areevaluated 24 hours after transfer of drug-treated cells to drug-freemedium. The endpoints used for MIC determination are either 100%inhibition (Tables 1 and 4) or (as is usual) 80% inhibition (Tables 2and 3) (Terb=terbinafine; Flu=fluconazole; Itra=itraconazole;C.=Candida).

                  TABLE 1                                                         ______________________________________                                        In vitro testing of the combination                                           terbinafine/fluconazole against fluconazole-resistant                         strains of Candida albicans, C. glabrata, C. krusei and C. tropicalis.                 MIC for 100 % inhibition (μg/ml)                                           Alone            Combination                                         Strain     Terb   Flu         Terb  Flu                                       ______________________________________                                        C. albicans                                                                              >100   >100        12.5  6.25                                      C. albicans                                                                                 >100                                                                                    >100        12.5                                                                                12.5                                C. albicans                                                                                 >100                                                                                    >100        12.5                                                                                6.25                                C. albicans                                                                                 >100                                                                                    >100        3.13                                                                                12.5                                C. tropicalis                                                                             >100        >100        >100                                                                             >100                                   C. glabrata                                                                                 >100                                                                                    >100        3.13                                                                                25.0                                C. albicans                                                                                 >100                                                                                    >100        3.13                                                                                50.0                                C. krusei       >100                                                                                  50.0              50.0                                ______________________________________                                    

In contrast to exposure of fungi to fluconazole or terbinafine alone,fungal growth was 100% inhibited in all but one strain when combinationsof fluconazole and terbinafine were used. In four of the strains,combinations of both drugs at concentrations <100 μg/ml were fungicidalin action, a result which could not be achieved with either of the drugsalone.

                  TABLE 2                                                         ______________________________________                                        In vitro testing of the combination                                           terbinafine/fluconazole against fluconazole-resistant Candida isolates                 MIC for 80% inhibition (μg/ml)                                             Alone           Combination                                          Strain     Flu    Terb       Flu   Terb                                       ______________________________________                                        C. albicans                                                                              >16    >1         0.25  0.1                                        C. albicans                                                                                >16        >1       16                                                                                     0.5                                 C. tropicalis                                                                            >16          >1       >0.125                                                                          0.06                                       C. glabrata                                                                                >16        >1       16                                                                                     0.125                               ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        In vitro testing of the combination                                           terbinafine/itraconazole against itraconazole-resistant Candida isolates                MIC for 80% inhibition (μg/ml)                                             Alone           Combination                                         Strain      Itra   Terb       Itra  Terb                                      ______________________________________                                        C. albicans >4     >1         ≦0.03                                                                        0.03                                      C. tropicalis                                                                              >4         ≦0.03                                                                         >1      0.03                                   ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        In vitro testing af the combination                                           terbinafine/compound A against fluconazole-resistant Candida isalates                MIC for 100 % inhibition (μg/ml)                                           Alone         Combination                                              Strain   Compound A  Terb    Compound A                                                                              Terb                                   ______________________________________                                        C. albicans                                                                            >100        >100    0.4       3.13                                   C. albicans                                                                               100                  0.4           3.13                           C. aibicans                                                                               100                  1.56         3.13                            C. albicans                                                                               100                  0.8           3.13                           ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        Summary of azole-resistant fungal strains in which a                          combination of terbinafine and an azole was found active and                  the azole to which resistance occurred                                                                 Proportion of                                                                 strains showing                                      Fungal species                                                                              Azole      activity                                             ______________________________________                                        C. albicans   Fluconazole                                                                               5 of 10                                             C. albicans            Itraconazole                                                                            9 of 10                                      C. tropicalis        Fluconazole                                                                               2 of 2                                       C. krusei                Fluconazole                                                                           1 of 4                                       C. paratropicalis                                                                              Fluconazole                                                                                   1 of 1                                       C. glabrata            Fluconazole                                                                             1 of 2                                       Cryptococcus          Fluconazole                                                                               3 of 10                                     neoformans                                                                    C.albicans                       4 of 4                                       ______________________________________                                    

We claim:
 1. A method of treatment of an azole-resistant fungalinfection caused by azole-resistant fungal strains, comprisingadministering a therapeutically effective amount of terbinafine offormula I ##STR2## in free base or acid addition salt form, and an azole14α-methyldemethylase inhibitor, to a patient in need of such treatment.2. A method according to claim 1 wherein terbinafine is in the form of ahydrochloride salt.
 3. A method according to claim 1 wherein the azoleis fluconazole.
 4. A method according to claim 1 wherein the azole isitraconazole.
 5. A method according to claim 1 wherein the azole is(R)-(-)-α-(4-chlorophenyl)-α-(1-cyclopropyl-1-methylethyl)-1H-1,2,4-triazol-1-ethanolin free form or in salt or metal complex form.
 6. A method according toclaim 1 wherein the azole-resistant fungal strain is a yeast strain. 7.A method according to claim 1 wherein the azole-resistant fungal strainis a Candida strain.
 8. A method according to claim 1 wherein the azoleresistant fungal strain is Candida albicans.
 9. A method according claim1 wherein the azole-resistant fungal strain is a Cryptococcus strain.10. A method according to claim 1 wherein the terbinafine and azole areadministered together.
 11. A method according to claim 1 wherein theterbinafine and azole are administered separately.
 12. A method ofpreventing azole resistance to a fungal infection susceptible toacquiring azole-resistance comprising administering a therapeuticallyeffective amount of terbinafine of formula I ##STR3## in free base oracid addition salt form, and an azole 14α-methyldemethylase inhibitor,to a patient having a fungal infection susceptible to acquiringazole-resistance.
 13. A method according to claim 12 wherein terbinafineis in the form of a hydrochloride salt.
 14. A method according to claim12 wherein the azole is fluconazole.
 15. A method according to claim 12wherein the azole is itraconazole.
 16. A method according to claim 12wherein the azole is(R)-(-)-α-(4-chlorophenyl)-α-(1-cyclopropyl-1-methylethyl)-1H-1,2,4-triazol-1-ethanolin free form or in salt or metal complex form.
 17. A method according toclaim 12 wherein the fungal infection is caused by a fungal strainsusceptible to acquiring azole resistance.
 18. A method according toclaim 12 wherein the fungal strain is a yeast strain.
 19. A methodaccording to claim 12 wherein the terbinafine and azole are administeredtogether.
 20. A method according to claim 12 wherein the terbinafine andazole are administered separately.